Infectious salmon anemia (ISA) is a disease that mainly affects Atlantic salmon (Salmo salar) causing huge losses in salmon farming worldwide (1, 14). The clinical signs of this disease are the presence of pale gills, ascites, hemorrhagic necrosis of the liver, splenomegaly, congestive gut and acute anemia (14). ISA was first reported in Norway in 1984 (28), later diagnosed in Canada (4), Scotland (27), United States (18), Faroe Islands (18) and Chile (16).
The etiological agent of this disease is a packaged pleomorphic virus between 45 and 140 nm of diameter, called Infectious Salmon Anemia Virus (ISAV), belonging to the Orthomixoviridae family (8). Its genome consists of 8 ssRNA of negative polarity (ns-RNA) that encode for 10 proteins and which have Untranslated Regions (UTRs) at both ends (26).
There is a limited knowledge about the functions of each ISAV protein performs. Bioinformatics evidence indicates that segments 1, 2 and 4 would encode for dependent RNA polymerase subunits (RpRd), analogous to PB2, PB1 and PA of Influenza A virus, respectively. Segment 3 encodes for NP protein, which has been reported to have the ability to bind ssRNA (2). As for Influenza, it has been proven that these four polypeptides relate to each one of the eight viral RNA segments to form Ribonucleoprotein complexes (RNPs) (23). These eight RNP units correspond to the minimum infectious unit required to initiate a cell infection (23). Segment 5 encodes for the Fusion protein (F), which has shown to be present on the surface of the viral membrane, allowing at the first steps of the infection, the fusion of the membrane of the viral particle with the cell endosome, allowing the release of the RNPs into cell cytoplasm (3). Segment 6 encodes for hemagglutinin-esterase protein (HE), which is also present on the viral surface and whose function is to bind a sialic acid residue located in the cell receptor (15). HE protein also has receptor-destroying activity (RDE, receptor destroying enzyme), favoring the release of new viral particles emerging from the cell membrane (21). Contrary to what is observed in hemagglutinin from Influenza A virus, whose stalk region is highly kept, it has been reported that ISAV's HE protein has a highly variable region towards the carboxy terminal end and adjacent to the transmembrane region, also known as Highly Polymorphic Region (HPR) (9). This HPR region encodes for 35 amino acids and, based on its high polymorphism 30 variants have been described in Europe, North America and Chile (30, 31, 32). One theory explains this variation as a deletion phenomenon from an ancestral strain present in the longest HPR region that is called HPRO (33). The first HPRO strain was identified in Scotland in wild salmon that did not show any clinical signs of ISA, being classified as an avirulent strain (34). In contrast, those strains presenting deletions in that zone are capable of developing virulent ISA-related clinical signs and mortality. It is suggested that segment 7 encodes for non-structural proteins analogous to NS1 and NS2 of influenza A virus (19). Finally, segment 8 encodes for a transcript containing two overlapping open reading frames (ORF, Open reading Frame). ORF1 encodes for the matrix protein (M), and ORF2 encodes for M2 protein. It has been shown that M2 protein is involved in the modulation of the type-I IFN response in conjunction with the NS1 protein (12).
As regards the Influenza Virus, detailed study of the virus has been possible as a result of the development of a reverse genetics system, which allows to manipulate the virus genome, being able to determine possible causes of virulence, as well as detailed study of each one of the functions of viral proteins (13). The most widely used reverse genetics system on Influenza virus is the plasmid-based system which allows to generate recombinant viruses from cloned cDNA. ISA Virus has 8 genomic RNAs transcribed under control of RNA polymerase I and the proteins making up the ribonucleoprotein complex under the command of RNA polymerase II are expressed (10, 24).
At the date there are no reports describing a successful reverse genetics system on ISAV. A relevant difficulty in generating a reverse genetics system is having the defined promoter for RNA polymerase I, which has not yet been described for Atlantic salmon. The difficulty lies in that promoters for RNA Polymerase I are strictly species-specific, they do not have a clear genetic structure and are in the IGS region of rDNA, which are vast (6). Identification of the sequences corresponding to the promoter for Pol I and its enhancers is hard work, considering that the IGS in the Salmo gender varies between 15-23 kb length (5). For this reason, and in view of the need of having a promoter with RNA Pol I characteristics, here we evaluate the capacity of the 571 pb ITS-1 region (Internal Transcribed Sequences) as previously described for Salmo salar (Atlantic salmon) (25). It has been recently described in nematodes, through bioinformatics analysis, that the ITS-1 region contains transcription promoter motifs and regulator motifs in which their function are not been demonstrated yet (29). It is suggested in this study that in the ITS-1 region of the rDNA, there are motifs having promoter characteristics and transcription regulators that have been conserved for millions of year of evolution, differing between species of the same gender, although they suggest the making of in vitro transcription assays to prove it.
The present invention shows that ITS-1 region of the rDNA of Atlantic salmon shows transcription promoter activity, which has conserved for millions of years; however, to confirm this assertion in vitro transcription essays had to be made.
In Chile and all other salmon farmers, there is the urgent need of figure out virulence factors, pathogenesis mechanisms of ISA virus, and an efficient vaccine against the only member of the Isavirus gender, the implementation of a plasmidal reverse genetics system allowing to generate recombinant ISA virus (ISAVr) becomes a necessity. With this goal, the challenge of developing a reverse genetics system for ISAV based on plasmids and using innovative elements, such as the use of salmon's ITS-1 region which has never been described as a promoter element. A choice that turned out to be key for the success of the systems that will be described below.